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The goal of this study was to apply the principles of analytical quality by design (AQbD) to the analytical method for determining the radiochemical purity (PQR) of the radiopharmaceutical sodium iodide 131I oral solution, utilizing thin-layer chromatography (TLC) with a radio-TLC scanner, which also enables the evaluation of product quality. For AQbD, the analytical target profile (ATP), critical quality attributes (CQA), risk management, and the method operable design region (MODR) were defined through response surface methodology to optimize the method using MINITAB® 19 software. This study encompassed the establishment of a control strategy and the validation of the method, including the assessment of selectivity, linearity, precision, robustness, detection limit, quantification limit, range, and the stability of the sample solution. Under the experimental conditions, the method parameters of the TLC scanner were experimentally demonstrated and optimized with an injection volume of 3 µL, a radioactive concentration of 10 mCi/mL, and a carrier volume of 40 µL. Statistical analysis confirmed the method's selectivity for the 131I iodide band Rf of 0.8, a radiochemical impurity IO3- Rf of 0.6, a linearity from 6.0 to 22.0 mCi/mL, and an intermediate precision with a global relative standard deviation (RSD) of 0.624%. The method also exhibited robustness, with a global RSD of 0.101%, a detection limit of 0.09 mCi/mL, and a quantification limit of 0.53 Ci/mL, meeting the prescribed range and displaying stability over time (at 0, 2, and 20 h) with a global RSD of 0.362%, resulting in consistent outcomes. The development of a method based on AQbD facilitated the creation of a design space and an operational space, with comprehensive knowledge of the method's characteristics and limitations. Additionally, throughout all operations, compliance with the acceptance criteria was verified. The method's validity was confirmed under the established conditions, making it suitable for use in the manufacturing process of sodium iodide 131I and application in nuclear medicine services.

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INTRODUCTION: Type 2 diabetes mellitus is a globally prevalent chronic disease characterised by hyperglycaemia and oxidative stress. The search for new natural bioactive compounds that contribute to controlling this condition and the application of analytical methodologies that facilitate rapid detection and identification are important challenges for science. Annona cherimola Mill. is an important source of aporphine alkaloids with many bioactivities. OBJECTIVE: The aim of this study is to isolate and identify antidiabetic compounds from alkaloid extracts with α-glucosidase and α-amylase inhibitory activity from A. cherimola Mill. leaves using an effect-directed analysis by thin-layer chromatography (TLC)-bioautography. METHODOLOGY: Guided fractionation for α-glucosidase and α-amylase inhibitors in leaf extracts was done using TLC-bioassays. The micro-preparative TLC was used to isolate the active compounds, and the identification was performed by mass spectrometry associated with web-based molecular networks. Additionally, in vitro estimation of the inhibitory activity and antioxidant capacity was performed in the isolated compounds. RESULTS: Five alkaloids (liriodenine, dicentrinone, N-methylnuciferine, anonaine, and moupinamide) and two non-alkaloid compounds (3-methoxybenzenepropanoic acid and methylferulate) with inhibitory activity were isolated and identified using a combination of simple methodologies. Anonaine, moupinamide, and methylferulate showed promising results with an outstanding inhibitory activity against both enzymes and antioxidant capacity that could contribute to controlling redox imbalance. CONCLUSIONS: These high-throughput methodologies enabled a rapid isolation and identification of seven compounds with potential antidiabetic activity. To our knowledge, the estimated inhibitory activity of dicentrinone, N-methylnuciferine, and anonaine against α-glucosidase and α-amylase is reported here for the first time.

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Mexico is considered one of the main regions of diversification of the genus Quercus (oaks). Oak species are one of the most important tree groups, particularly in temperate forests, due to its diversity and abundance. Some studies have shown that oak contains specialized metabolites with medicinal importance. In this work, the acetonic extract from leaves of three Mexican oaks (Quercus rugosa, Q. glabrescens, and Q. obtusata) was separated using thin-layer chromatography and column chromatography. Chemical identification of the major compounds was determined using high-performance liquid chromatography and nuclear magnetic resonance. Nineteen compounds were identified, three belonging to the terpenoid family (ursolic acid, ß-amyrin, and ß-sitosterol) and 16 from the phenolic family. Of the isolated compounds, seven are new reports for oak species (scopoletin, ursolic acid, ß-amyrin, luteolin-7-O-glucoside, kaempferol-3-O-sophoroside, kaempferol-3-O-glucoside, and kaempferol-3-O-sambubioside). More compounds were identified in Q. rugosa followed by Q. glabrescens and then Q. obtusata. The characterization of specialized metabolites in oak species is relevant, from both phytocentric and anthropocentric perspectives.

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The thin-layer chromatography technique (TLC) is a simple and inexpensive analysis commonly used to identify qualitatively the presence of carbohydrates in food samples such as mono- di and oligosaccharides particularly. TLC assay could be improved using image processing software for the semiquantitative determination of this type of compound. In the present work, TLC-image analysis with Silica Gel 60 TLC plates was used for the semiquantitative determination of 6 standards of carbohydrates (glucose, fructose, sucrose, 1-kestose, nystose, and fructofuranosylnystose). Subsequently, the areas of the spots of each compound were determined by digitizing in a conventional office scanner. Then, the segmentation of the images is carried out using software for image processing. The calibration curves were plotted in the Excel software using the average of the areas of the pigmentations obtained in pixels. In this study, the technique of thin-layer chromatography was also used to quantitatively determine the presence of carbohydrates in food samples such as honey, garlic, and onion. Values of determination coefficient (R2) greater than 0.97 in all the calibration curves were obtained. This technique could be useful for detecting carbohydrates (monosaccharides, disaccharides, and oligosaccharides) in analytical assays and food samples without needing specialized analytical equipment. In this work, it was possible to determine the concentration of carbohydrates in samples of garlic and onion that showed the presence of prebiotic carbohydrates in addition to sucrose, glucose, and fructose.

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Pepper is one of the most consumed spices in the world. Its main compound is capsaicin, a widely studied biomarker that has pharmacological activities due to its anti-inflammatory and analgesic capacity. Topical formulations such as patches based on capsaicin have been developed as an option in relieving pain and reducing swelling. In addition, capsaicin is used as an active ingredient in non-lethal weapon formulations such as pepper spray through the QuEChERS concept (Quick, Easy, Cheap, Effective, Robust, Safe) technique. Used for food analysis, it allows the direct analysis of the biomarker by TLC-ESI-MS, which are previously separated by HPTLC (High Performance Thin Layer Chromatography) using an internal standard for determination of Rf and confirmation of capsaicin in different matrices.[Formula: see text].

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Abstract Two sensitive and selective methods were developed for the simultaneous determination of four commonly used non-steroidal anti-inflammatory drugs (NSAIDs), namely; paracetamol (PCM), diclofenac sodium (DCF), ibuprofen (IBP), and indomethacin (IND) in wastewater effluents. The first method used HPLC for the determination of the studied drugs using a mobile phase consisting of phosphate buffer (pH 3.0) and acetonitrile at a flow rate of 1 mL/min. in gradient elution mode and detection at 220 nm. The separation process was performed on BDS Hypersil Cyano column (250 x 4.6 mm, 5 µm). The second method was a TLC-densitometric one which was performed using n-Hexane: ethyl acetate: acetic acid in the ratio (6:3.5:0.5) as a developing system. The proposed chromatographic methods were successfully applied for the selective determination of the four studied drugs in simulated and real pharmaceutical wastewater samples after their solid-phase extraction

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Eleutherine plicata has been shown to be a promising medicinal plant, and its activity has been associated with naphthoquinones. The present study aimed at evaluating the cytotoxicity, genotoxicity, and oral toxicity of the ethanol extract (EEEp), dichloromethane fraction (FDMEp) of E. plicata, and isoeleutherin. For the cytotoxicity evaluation, the viability test (MTT) was used. Genotoxicity was accessed through the Comet assay (alkaline version), acute and subacute oral toxicities were also evaluated. The antioxidant capacity of the samples in the wells where the cells were treated with E. plicata was evaluated. Furthermore, the participation of caspase-8 in the possible mechanism of action of isoeleutherin, eleutherin, and eleutherol was also investigated through a docking study. FDMEp and isoeleutherin were cytotoxic, with higher rates of DNA fragmentation observed for FDMEp and isoeleutherin, and all samples displayed higher antioxidant potential than the control. In the acute oral toxicity test, EEEp, FDMEp, and isoeleutherin did not cause significant clinical changes. In the subacute toxicity assay, EEEp and FDMEp also did not cause clinical, hematological, or biochemical changes. The three compounds bound similarly to caspase-8. Despite the results of cytotoxicity, in vitro studies demonstrated that the use of EEEp appears to be safe and cell death may involve its binding to caspase-8.

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Trypanosoma cruzi is the agent of Chagas disease, an infection that affects around 8 million people worldwide. The search for new anti-T. cruzi drugs are relevant, mainly because the treatment of this disease is limited to two drugs. The objective of this study was to investigate the trypanocidal and cytotoxic activity and elucidate the chemical profile of extracts from the roots of the Lonchocarpus cultratus. Roots from L. cultratus were submitted to successive extractions with hexane, dichloromethane, and methanol, resulting in LCH, LCD, and LCM extracts, respectively. Characterization of extracts was done using 1H-RMN, 13C-RMN, CC and TLC. Treatment of T. cruzi forms (epimastigotes, trypomastigotes, and amastigotes) with crescent concentrations of LCH, LCD, and LCM was done for 72, 48, and 48 h, respectively. After this, the percentage of inhibition and IC50/LC50 were calculated. Benznidazole was used as a positive control. Murine macrophages were treated with different concentrations of both extracts for 48 h, and after, the cellular viability was determined by the MTT method and CC50 was calculated. The chalcones derricin and lonchocarpine were identified in the hexane extract, and for the first time in the genus Lonchocarpus, the presence of a dihydrolonchocarpine derivative was observed. Other chalcones such as isocordoin and erioschalcone B were detected in the dichloromethane extract. The dichloromethane extract showed higher activity against all tested forms of T. cruzi than the other two extracts, with IC50 values of 10.98, 2.42, and 0.83 µg/mL, respectively; these values are very close to those of benznidazole. Although the dichloromethane extract presented a cytotoxic effect against mammalian cells, it showed selectivity against amastigotes. The methanolic extract showed the lowest anti-T. cruzi activity but was non-toxic to peritoneal murine macrophages. Thus, the genus Lonchocarpus had demonstrated in the past action against epimastigotes forms of T. cruzi but is the first time that the activity against infective forms is showed, which leading to further studies with in vivo tests.

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The objective of this study was to conduct phytochemical characterization and biological evaluation of Hymenaea eriogyne. Crude extracts and fractions from the bark, leaves and pods, were obtained for phytochemical screening by TLC and HPLC, and evaluation of antibacterial and antioxidant potential. Chromatographic data revealed the presence of several metabolites, notably from the flavonoid class. HPLC analysis confirmed the presence of the flavanonol astilbin (taxifolin 3-O-ramnoside) and other flavonoids derived from aglycone taxifolin. In addition, it was possible to quantify phytochemical markers in the extracts and fractions, which showed an increased content of flavonoid and catechin derivatives in the fraction. Better results of the minimum inhibitory/bactericidal concentrations were obtained with extracts and fractions from bark. In the antioxidant activity using the DPPH method, the enriched bark fraction presented an IC50 of 34.46 µg/mL. These results contribute to the continuity of studies on the chemical and biological composition of the species.

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Cannabis sativa is the drug of abuse most cultivated, trafficked, and consumed worldwide. One of several techniques used to detect cannabinoids is based on the thin-layer chromatography (TLC). However, the designation of the colors observed can be inaccurate and not reproducible. The designation of colors goes beyond physical and physiological aspects, because what is conventionally called color is a socio-cultural construction. Thus, the objective of this paper was to evaluate the different TLC methods to detection of cannabinoids, and apply standardization method in naming of colors. TLC analysis performed using silica gel 60 F254 as a stationary phase. Three mobile phase compositions [hexane:chloroform (8:2 v:v), hexane:ethyl ether (8:2 v:v), and chloroform:hexane (8:2 v:v)], as well as, two different solutions of Fast Blue B salt (FBBS, Azoic Diazo No. 48) and Fast Blue RR (FBRR, Azoic Diazo No. 24) were evaluated. Determination of colors names was realized through the Sci-Chromus® software. The best resolution was obtained using hexane:ethyl ether (8:2 v:v) as a mobile phase. It was observed that although the cannabidiol (CBD), delta-9-tetrahydrocannabinol (Δ9 -THC), cannabinol (CBN), and cannabigerol (CBG) were detect using both the FBBS- and FBRR-acidified solutions, the best visualization was achieved using the latter reagent. To the best of our knowledge, this is the first study that applied and demonstrated a method for standardization and denomination of colors in the TLC analysis of cannabinoids. This method was able to reduce the subjectivity in naming the colors observed and presented several application possibilities.

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Abstract The objective of this study was to identify the emetic metabolites in different parts of the P. ipecacuanha, a plant with emetic properties. Partial phytochemical analysis was performed to determine the presence of emetine and cephaeline in leaves, stems and roots. Both alkaloids were detected in the three plant parts analyzed. Highest alkaloid content was found in roots (8.55 mg/g), followed by stems (4.05 mg/g), and the lowest was found in leaves (2.4 mg/g). The cephaeline content (8.35 mg/g) was higher than that of emetine (6.65 mg/g) in all the three organs analyzed. Toxicity analysis of the crude extract showed a LD50 of 500 mg/kg.

Resumen El objetivo de este estudio fue identificar los metabolitos eméticos en diferentes partes de P.ipecacuanha, una planta con propiedades eméticas. Se realizó un análisis fitoquímico parcial, donde se determinó la presencia de emetina y cefalina en hojas, tallos y raíces. Ambos alcaloides se detectaron en las tres partes de la planta analizadas. El mayor contenido de alcaloides se encontró en las raíces (8.55 mg/g), seguido de los tallos (4.05 mg /g), y el más bajo se encontró en las hojas (2.4 mg/g). El contenido de cefalina (8,35 mg/g) fue mayor que el de emetina (6,65 mg/g) en los tres órganos analizados. El análisis de toxicidad del extracto crudo mostró una DL50 de 500 mg/kg.

Resumo O objetivo deste estudo foi identificar os metabólitos eméticos em diferentes partes do P. ipecacuanha, uma planta com propriedades eméticas. Foi realizada análise fitoquímica parcial para determinar a presença de emetina e cefelina nas folhas, caules e raízes. Ambos os alcalóides foram detectados nas três partes da planta analisadas. O maior teor de alcalóides foi encontrado nas raízes (8,55 mg/g), seguido dos caules (4,05 mg/g), e o menor foi encontrado nas folhas (2,4 mg/g). O conteúdo de cefhaelina (8,35 mg/g) foi superior ao de emetina (6,65 mg/g) nos três órgãos analisados. A análise de toxicidade do extrato bruto mostrou um LD50 de 500 mg/kg.

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RESUMEN El objetivo del presente artículo es determinar cómo se vieron afectadas las condiciones laborales del sector agropecuario colombiano, dentro del marco de efectos sociales, tras la firma del acuerdo de promoción comercial entre Colombia y Estados Unidos. Para esto, se realizó una adaptación macroeconómica de los indicadores GRI versión G4 en la categoría desempeño social. Esta adaptación permitió analizar la información sectorial y aplicarlos en la población laboral del sector agropecuario colombiano. La metodología utilizada fue cualitativa con énfasis en un método de lista de comprobación y análisis de indicadores, y se encontraron dos resultados importantes. En primer lugar, que el acuerdo comercial entre ambos países es superficial al momento de salvaguardar las condiciones laborales del sector, sin profundizar en aspectos específicos que reglamenten el contexto laboral. En segundo lugar, se evidenció que a pesar de los aumentos significativos en las tasas de contrataciones, las afiliaciones a entidades prestadoras de salud o administradoras de riesgos profesionales de empleados, la mayor parte del sector agropecuario colombiano opera bajo la informalidad. Finalmente, se puede afirmar que las condiciones laborales del sector agropecuario colombiano, dentro del marco de efectos sociales, tras la firma del acuerdo de promoción comercial entre Colombia y Estados Unidos no se vieron afectadas de manera positiva. La informalidad y la falta de regulación sigue siendo un factor común en el sector.

ABSTRACT The objective of this article is to determine how the labor conditions of the Colombian agricultural sector were affected, within the framework of social effects, after the signing of the Trade Promotion Agreement between Colombia and the United States. For this, a macroeconomic adaptation of GRI indicators version G4 in the social performance category was made. This adaptation made it possible to analyze sectoral information and apply it to the labor population of the Colombian agricultural sector. The methodology used was qualitative with emphasis on a checklist method and analysis of indicators, and two important results were found. First, that the trade agreement between both countries is superficial at the moment of safeguarding the labor conditions of the sector, without going into specific aspects that regulate the labor context. Secondly, it was evident that despite the significant increases in contracting rates, affiliations with health providers or managers of occupational risks for employees, most of the Colombian agricultural sector operates under informality. Finally, it can be affirmed that the labor conditions of the Colombian agricultural sector, within the framework of social effects, after the signing of the Trade Promotion Agreement between Colombia and the United States were not affected in a positive manner. Informality and lack of regulation continues to be a common factor in the sector.

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Neurolaena lobata (L.) R.Br. ex Cass. (Asteraceae)Is a popular folk remedy for in Central America. The plant is of commercial value in Guatemala but so far there is not any monograph to guide regional laboratories on ensuring identity and chemical tests for this species. As identity test we here run macro and micro morphoanatomical studies of the characters of the vegetative organs. We also developed standard chemical tests for quality by both TLC and HPLC for infusions and tinctures of varying alcoholic strength. Their radical scavenging activities in DPPH and NO were also measured. Macro and micro morphoanatomical characters of the vegetative organs present a set of characteristics to facilitate the identification of dry powdered samples of this species. We developed optimal conditions for the TLC and HPLC phytochemical fingerprints of the 4 most common pharmacopoeial liquid herbal preparations from this herbal drug, namely infusion, 70%, 45% and 20% hydroalcoholic tinctures. Our work provides the Latin-American industry with a set of analyses to establish the identity and chemistry of N. lobata samples for quality control purposes.

Neurolaena lobata (L.) R.Br. ex cass. (Asteraceae) es un remedio popular popular en América Central. La planta tiene un valor comercial en Guatemala, pero hasta el momento no existe una monografía que guíe a los laboratorios regionales para garantizar la identidad y las pruebas químicas para esta especie. Como prueba de identidad proponemos estudios macro y micro morfoanatómicos de los caracteres de los órganos vegetativos. También desarrollamos pruebas químicas de calidad mediante CCF y CLAR para infusiones y tinturas de grado alcohólico variable. También se midieron sus actividades de captación de radicales en DPPH y NO. Los caracteres macro y micro morfoanatómicos de los órganos vegetativos presentan un conjunto de características para facilitar la identificación de muestras de polvo seco de esta especie. Desarrollamos condiciones óptimas para las huellas dactilares fitoquímicas de CCF y CLAR de las 4 preparaciones herbales líquidas farmacopéicas más comunes de esta droga herbal, a saber, infusión, 70%, 45% y 20% tinturas hidroalcohólicas. Nuestro trabajo proporciona a la industria latinoamericana un conjunto de análisis base para establecer la identidad y la química de las muestras de N. lobata con fines de control de calidad.

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Cancer is one of the biggest problems in public health worldwide. Plants have been shown important role in anticancer research. Viscum album L. (Santalaceae), commonly known as mistletoe, is a semi-parasitic plant that grows on different host trees. In complementary medicine, extracts from European mistletoe (Viscum album L.) have been used in the treatment of cancer. The study was conducted to identify chemical composition and antitumor potential of Viscum album tinctures. Chemical analysis performed by high resolution chromatography equipped with high resolution mass spectrometer identified caffeic acid, chlorogenic acid, sakuranetin, isosakuranetin, syringenin 4-O-glucoside, syringenin 4-O-apiosyl-glucoside, alangilignoside C and ligalbumoside A compounds. Some of these compounds are probably responsible for the reduction of tumoral cellular growth in a dose-dependent manner. It was observed that melanoma murine cells (B16F10) were more sensitive to V. album tinctures than human leukaemic cells (K562), besides non-tumoral cells (MA-104) had a much lower cytotoxicity to them. Apoptotic-like cells were observed under light microscopy and were confirmed by a typical DNA fragmentation pattern. Additionally, flow cytometry results using Annexin-V/FITC permitted to quantify increased expression of early and late apoptotic markers on tumoral cells, confirming augmented Sub G0 population, which was probably associated with a consistent decrease in G1, and an increase in S or G2/M populations. Results indicate the chemical composition of V. album tinctures influences the mechanisms of in vitro tumoral cell death, suggesting a potential use in cancer pharmacotherapy research.

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The objective of this study was to measure the effects of glucose and salt level on white blood cells, red blood cells and platelets (PLTs) in the blood of a leukemic patient by using a white light microscope. Different concentrations of glucose and salt in the range of 0 mM to 500 mM were admixed in the blood sample to prepare blood smear. We revealed that shape of erythrocytes, leukocytes and platelets changes and form aggregates. Increasing concentrations of glucose cause to increases aggregation process of white blood cells, red blood cells and platelets. And the increasing concentration of sodium chloride causes to increase rouleaux formation and aggregation of platelets but dehydration due to increased sodium chloride concentration causes to break the aggregation of white blood cells. Comparison of CBC reports of these samples with and without analytes shows that total leukocyte count (TLC) decreases gradually towards normal ranges of leukocytes which is favorable in the treatment of leukemia but at the same time decreasing level of hemoglobin HGB, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and increasing level of red blood cell (RBCs) causes to reduce oxygen supply which is in favor of cancer growth and anemia. This work provides us the base for translation this in vitro study towards the in vivo case of blood microvasculature as a non-invasive methodology.

O objetivo deste estudo foi medir os efeitos da glicose e do nível de sal nos glóbulos brancos, glóbulos vermelhos e plaquetas (PLTs) no sangue de um paciente leucêmico usando um microscópio de luz branca. Foram misturadas diferentes concentrações de glicose e sal na gama de 0 mM a 500 mM na amostra de sangue para preparar esfregaço de sangue. Descrevemos que a forma dos eritrócitos, leucócitos e plaquetas muda e forma agregados. O aumento das concentrações de glicose aumenta o processo de agregação de glóbulos brancos, glóbulos vermelhos e plaquetas. E a crescente concentração de cloreto de sódio causa o aumento da formação de rouleaux e a agregação de plaquetas, mas a desidratação devido ao aumento da concentração de cloreto de sódio causa a quebra da agregação de glóbulos brancos. A comparação dos relatórios de CBC dessas amostras com e sem analitos mostra que a contagem total de leucócitos (TLC) diminui gradualmente para os intervalos normais de leucócitos, o que é favorável no tratamento da leucemia, mas ao mesmo tempo diminui o nível de hemoglobina HGB, hemoglobina corpuscular média (MCH ), a concentração média de hemoglobina corpuscular (MCHC) e o aumento do nível de glóbulos vermelhos (RBCs) reduz o suprimento de oxigênio, o que é a favor do crescimento do câncer e da anemia. Este trabalho fornece a base para a tradução deste estudo in vitro para o caso in vivo de microvasculatura de sangue como uma metodologia não-invasiva.

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Forced degradation studies of gliquidone were conducted under different stress conditions. Three degradates were observed upon using HPLC and TLC and elucidated by LC-MS and IR. HPLC method was performed on C18 column using methanol-water (85:15 v/v) pH 3.5 as a mobile phase with isocratic mode at 1 mL.min-1 and detection at 225 nm. HPLC analysis was applied in range of 0.5-20 µg.mL-1 (r =1) with limit of detection (LOD) 0.177 µg.mL-1. TLC method was based on the separation of gliquidone from degradation products on silica gel TLC F254 plates using chloroform-cyclohexane-glacial acetic acid (6:3:1v/v) as a developing system with relative retardation 1.15±0.01. Densitometric measurements were achieved in range of 2 -20 µg /band at 254 nm (r = 0.9999) with LOD of 0.26 µg /band. Least squares regression analysis was applied to provide mathematical estimates of the degree of linearity. The analysis revealed a linear calibration for HPLC where a binomial relationship for TLC. Stability testing and methods validation have been evaluated according to International Conference on Harmonization guidelines. Moreover, the proposed methods were applied for the analysis of tablets and the results obtained were statistically compared with those of pharmacopeial method revealing no significant difference about accuracy and precision.

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ABSTRACT The aim of this study is the production, purification, and characterisation of thermostable raw starch hydrolyzing α-amylase produced by Bacillus mojavensis SO-10. The maximum production conditions of α-amylase were found at 36th hour, 35 °C and pH 7.0. We utilized three steps to purify the thermostable α-amylase and as a result, 34-fold and 18% yield were obtained. The molecular weight of purified α-amylase was determined as 73 kD. The Km and Vmax rates were detected as 0.010 mM and 3.38 µmol min−1, respectively. This purified α-amylase exhibited the highest activity at pH 5.0-6.0 and 70 ºC and showed stability over a wide variety of pH and temperature at 4.0-8.0, and 40-50 ºC, respectively. The thermostable purified α-amylase exhibited stability in the presence of denaturing agents and heavy metal ions. The purified enzyme hydrolyzed the raw starches of corn and wheat grains in the ratio of 36.7% and 39.2% respectively. The end-yields of soluble starch hydrolysis were analyzed by thin-layer chromatography (TLC). In addition, the usage of purified α-amylase in clarification of apple juice and domestic washing detergent industries were evaluated.

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Due to inconsistency in demand and supply of fishmeal there is immense need of alternate protein sources. Present project was therefore designed to replace costly fishmeal (FM) with low-priced fermented fish silage (FFS) in fish feed. Fermented fish silage was prepared by fermentation process using Lacto bacillus bacteria and its fatty acid profile and effect on digestive system of Labeo rohita was investigated. Lipid contents were isolated by Soxhlet apparatus and recorded as 6.23 ± 1.23 g/100g of fermented fish silage (FFS). Fatty acid profile of extracted Lipids was determined by gas liquid chromatography (GLC), sufficient amount of unsaturated fatty acids were found with pattern mono unsaturated fatty acids (MUFA) > saturated fatty acids (SFA) >poly unsaturated fatty acids (PUFA). Three treatment diets containing 100% silage (T1), 75% silage (T2) and 50% silage (T3) were prepared by mixing it with soybean meal (SBM) and rice bran as co-ingredients while fermented fish silage was replaced by fishmeal in control diet (T0). The experiment was conducted in glass aquaria in triplicate. Fish growth parameters were recorded fortnightly while physicochemical parameters of water were recorded on daily basis. After completion of feeding trial, three fish were randomly dissected to excise out their intestines and determine activity for protease, amylase and lipase enzymes. Non-significant differences (P<0.05) were recorded in growth parameters and enzymatic activity among all diets except lipase enzyme. Deceptively, it can be concluded that FFS has reasonable concentration of nutrients and unsaturated fatty acids so it can successfully replace fishmeal in fish diets.

Devido à diferença na procura e na oferta de farinha de peixe há imensa necessidade de qualquer membro suplente da fonte de proteína. Tão presente projeto foi projetada para substituir a dispendiosa farinha de peixe (FM) com barato peixe fermentado de ensilagem (FFS) em alimentos para peixes. FFS foi preparado pelo processo de fermentação usando Lactobacillus bactérias e seu perfil de ácidos graxos e efeito sobre o sistema digestivo de Labeo rohita foi investigado. Conteúdo lipídico foram isoladas pelo aparelho de Soxhlet e registadas como 6,23 ± 1,23 g/100g de FFS. Perfil de ácidos graxos extraídos de lipídios foi determinada por cromatografia líquida de gás (GLC). Quantidade suficiente de ácidos graxos insaturados foram encontrados com padrão MUFA > SFA > AGPI. Tratamento de três dietas contendo silagem de 100% (T1), 75% silagem (T2) e 50% silagem (T3) foram preparados misturando com farinha de soja (SBM) e farelo de arroz como co ingredientes enquanto FFS foi substituído pela FM na dieta controle (T0). O experimento foi conduzido em aquários de vidro em triplicado. O Crescimento dos peixes foram anotados os parâmetros quinzenal enquanto parâmetros físico-químicos de água foram registradas diariamente. Após a conclusão do teste de alimentação, três peixes foram aleatoriamente dissecada a impostos especiais de consumo os seus intestinos e determinar a atividade de protease, enzimas amilase e lipase. As variações não significativas (P<0,05) foi registrada em parâmetros de crescimento e atividade enzimática entre as dietas exceto enzima lipase mostrou diferença significativa entre as dietas de tratamento. Aparentemente, é possível concluir que a concentração razoável de FFS tem nutrientes e ácidos graxos insaturados de modo que ela possa substituir com êxito a farinha de peixe na dieta de peixes.

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INTRODUCTION: Reverse phase chromatography and bioautographic assays are key tools for natural product bioguided isolation; however, their direct coupling has not been fully achieved. OBJECTIVES: To develop a bioautographic assay to detect tyrosinase inhibitors present in complex matrices sorbed on reverse phase (RP) TLC-plates that can be used for bioguided isolation of bioactive compounds. METHODS: Enzyme gel entrapment with an amphiphilic copolymer was used for assay development. The gel turns into a brown "skin like" colour due to tyrosinase catalysed oxidation of l-tyrosine. The inhibitors are visualised as clear spots against a brown coloured background. RESULTS: The assay was able to localise cinnamaldehyde in Cinnamomum cassia essential oil, as its main constituent with known tyrosinase inhibition properties. The assay allowed the detection of 0.03% (w/w) of kojic acid co-spotted with a methanolic extract of Sphaeralcea bonariensis and chromatographed on RP-TLC. CONCLUSION: The developed assay is able to detect, with high sensitivity, tyrosinase inhibitors present in complex matrices that were chromatographed in RP-TLC. Results can be easily read by colour change, inhibitors appear as clear spots in a darker background. Copyright © 2016 John Wiley & Sons, Ltd.

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Mono e diacilgliceróis são produtos empregados na indústria alimentícia, farmacêutica, cosmética e química como emulsificantes e melhoradores de viscosidade de produtos alimentícios, cosméticos e farmacêuticos. No entanto, a forma mais usual de obtê-los é por síntese química, o que acaba rendendo produtos finais caros e com atributos de qualidade, rendimento e de aplicabilidade tecnológica inferiores aos esperados. A busca por formas de obtenção mais racionais, eficientes e com melhor padrão de qualidade destes produtos foi o objetivo principal do trabalho, por meio de hidrólise parcial enzimática, que necessita de condições de reação mais brandas. Foram avaliadas a hidrólise enzimática descontína, empregando como substrato a trioleína técnica, e a hidrólise enzimática descontínua-alimentada, usando como substrato o óleo de girassol médio oléico. Foi utilizada, em ambos processsos, a lipase imobilizada sn-1,3 específica Lipozyme RM IM (de Rhizomucor miehei). A caracterização dos padrões e dos substrados, bem como o acompanhamento da formação dos produtos da hidrólise enzimática foram feitos por determinação da porcentagem de hidrólise, cromatografia em camada delgada (TLC), dos perfis das curvas de fusão e cristalização por calorimetria diferencial de varredura (DSC), cromatografia gasosa (CG) e cromatografia de exclusão de tamanho de alto desempenho (HPSEC). Os parâmetros de hidrólise descontínua foram o tempo de reação, a temperatura e a concentração inicial de substrato. Os parâmetros de hidrólise descontínua-alimentada foram tempo de enchimento e intervalo de alimentação de substrato. Para as respostas analíticas de porcentagem de hidrólise e de composição de frações lipídicas foi aplicado um modelo de regressão múltipla com base em metodologia de superfície de resposta. Os resultados experimentais observados nas reações de hidrólise enzimática descontínua de trioleína técnica mostraram de 24,7 a 34,2% de mono e diacilgliceróis (para 5% de óleo na emulsão) e de 21,4 a 33,6% de mono e diacilgliceróis (para 20% de óleo na emulsão). Os resultados experimentais observados nas reações de hidrólise enzimática descontínua-alimentada de óleo de girassol médio oléico (para 15% de óleo na emulsão), mostraram de 7,9 a 31,8% de mono e diacilgliceróis. Os modelos de superfície de resposta foram considerados significativos e preditivos. As hidrólises obtidas no formato descontínuo e descontínuo-alimentado permitiram efetivamente a obtenção de frações de mono/ diacilgliceróis com vários graus de eficiência de conversão e com corretas identificação e quantificação das frações de lipídios procuradas. As correlações feitas entre porcentagem de hidrólise e entalpias de cristalização e fusão, corroboradas com os resultados qualitativos e/ou quantitativos diretos obtidos na cromatografia de camada delgada (TLC) e de HPSEC, demonstraram que estes atributos podem positivamente indicar a ocorrência efetiva de reação de hidrólise, além de auferir uma escala de desempenho de reação alinhada com o previsto na literatura, à medida que são aumentadas a temperatura, o tempo de hidrólise e a porcentagem inicial de substrato oleoso, sob regime descontínuo, e que puderam ser melhoradas, de forma inovadora, sob parâmetros de tempo total de alimentação e de intervalo de alimentação, sob regime descontínuo-alimentado. A hidrólise parcial enzimática de triacilgliceróis utilizando lipase imobilizada sn-1,3 específica pode ser considerada uma alternativa às vias químicas para a produção de misturas de mono e diacilgliceróis para utilização como aditivos químicos.

Mono and diacylglycerols are products used in the food, pharmaceutical, cosmetic and chemical industries as emulsifiers and viscosity improvers for food products, cosmetics and pharmaceuticals. However, the most usual forms of obtaining them are by chemical synthesis, which ends up yielding expensive final products with attributes of quality, yield and technological applicability lower than expected. The search for more rational, efficient and better quality standards of these products was the aim of the work, through partial enzymatic hydrolysis, which requires milder reaction conditions. Discontinuous enzymatic hydrolysis was evaluated using technical triolein as substrate and discontinuous-fed enzymatic hydrolysis using as the substrate the mid oleic sunflower oil. In both processes, immobilized lipase sn-1,3 specific Lipozyme RM IM (from Rhizomucor miehei) was used. The characterization of the patterns and substrates, as well as the monitoring of the formation of the products from the enzymatic hydrolysis were made by determining the percentage of hydrolysis, thin layer chromatography (TLC), profiles of the melting and crystallization curves by differential scanning calorimetry ( DSC), gas chromatography (GC) and high performance size exclusion chromatography (HPSEC). The parameters of discontinuous hydrolysis were the reaction time, the temperature and the initial substrate concentration. The parameters of discontinuous-fed hydrolysis were filling time and substrate feed interval. For the analytical responses of hydrolysis percentage and composition of lipid fractions a multiple regression model was applied based on response surface methodology. The experimental results observed in the reactions of discontinuous enzymatic hydrolysis of technical triolein indicated amounts of mono- and diacylglycerols from 24.7 to 34.2% (for 5% of oil in the emulsion) and from 21.4 to 33.6% for mono and diacylglycerols with 20% oil in the emulsion. The experimental results observed in the reactions of discontinuous-fed enzymatic hydrolysis of mid oleic sunflower oil (for 15% oil in the emulsion), showed from 7.9 to 31.8% of mono and diacylglycerols. Response surface models were considered significant and viii predictive. The hydrolysis obtained in the discontinuous and discontinuous-fed form allowed to obtain fractions of mono / diacylglycerols with various degrees of conversion efficiency and with correct identification and quantification of the lipid fractions sought. The correlations between the percentage of hydrolysis and enthalpies of crystallization and fusion, corroborated with the qualitative and / or quantitative direct results obtained in thin layer chromatography (TLC) and HPSEC, showed that these attributes can positively indicate the effective occurrence of reaction of Hydrolysis, in addition to achieving a reaction performance scale in line with the literature, as the temperature rate, the hydrolysis time and the initial percentage of oily substrate are increased under a discontinuous regime and can be improved, in a innovative form, under parameters of total filling time and feeding interval, under a fed-batch regime. The partial enzymatic hydrolysis of triacylglycerols using specific sn-1,3-specific immobilized lipase may be considered an alternative to the chemical pathways for the production of mono- and diacylglycerol blends for use as chemical additives.

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